Regulation of extracellular glutamate in the neonatal brain is critical to prevent seizures that can have severe consequences for cognitive development. Astrocytic processes encapsulate excitatory synapses and remove glutamate from the perisynaptic and extracellular space by means of two glial specific Na+-dependent glutamate transporters (GLT and GLAST). The central hypothesis of this grant is that astrocytic glutamate (Glu) transport contributes significantly to glutamatergic neuro- transmission in the developing CNS. Specifically, we speculate that astrocytic Glu uptake aids the termination of the synaptic response, but additionally, we suggest that neuronal activity can lead to non-vesicular Glu release by astrocytes, contributing to pre-synaptic transmitter release. Since the extracellular space is very small, these interactions do not require large interstitial changes from Glu. We will test these hypotheses through patch-clamp recording sin hippocampal and cerebellar slices from P3-P30 rats. We will simultaneously record from a post-synaptic neuron and an astrocyte or Bergmann glial cell encapsulating the synapse from which the neuron receives glutamatergic input. We seek to demonstrate that presynaptic neuronal response. We also propose to show that depolarization of the astrocyte induces non-vesicular Glu release, which in turn can directly induce postsynaptic responses. We also will question whether, during development, Glu transporters are selectively targeted to astrocytic processes near glutamatergic synapses. We hypothesize that during synaptogenesis, transporters are specifically recruited towards glutamatergic synapses by transmitter released from active synapses. To test this hypothesis, we will immunohistochemically examine expression of Glu transporters at glutamatergic synapses identified by expression of the presynaptic proteins SAP90 or GKAP, and will use time-lapse video-microscopy to study clustering of Glu transporters near artificial glutamate release sites in vitro. These experiments will help to establish the contribution of astrocytic glutamate transport in modulation of Glu synapses during development.